BASIS OF ACTINOMYCIN ACTION, II. EFFECT OF ACTINOMYCIN ON THE NUCLEOSIDE TRIPHOSPHATE-INORGANIC PYROPHOSPHATE EXCHANGE

In a preceding paper,1 we have described the effect of actinomycin on the different synthetic reactions catalyzed by RNAt-polymerase, and the relationship between actinomycin action and the structure and base composition of DNA. This paper presents the results of experiments concerned with the effect of actinomycin on the nucleoside triphosphate-inorganic pyrophosphate exchange reaction. Materials and Methods.-The isolation of RNA-polymerase from Escherichia coli B and the assay conditions used during its purification were as described by Chamberlin and Berg.2 All reactions contained 4.1 ,g enzyme protein/0.25 ml. Incorporation of radioactive pyrophosphate (PP3I) into the terminal pyrophosphate position of nucleoside triphosphates ("exchange reaction") was determined as follows: pp3I, prepared according to Jones et al.,3 was incubated with the components of the standard assay mixture, in the absence of Mn++, at a concentration of 1.6 Mmoles/ml; MgCl2 was present at 8 Mmoles/ml. The reaction was stopped by chilling, adding a large excess of nonradioactive phosphate and pyrophosphate buffers, carrier bovine serum albumin and yeast RNA (2 mg each per reaction vessel) and trichloracetic acid (final concentration 5%7,). After centrifugation, the supernatant solution was treated with charcoal; the latter was washed four times with acetate buffer (0.1 M pH 4.5) containing phosphate and pyrophosphate (0.03 ill), transferred to a planchet, dried, and the adsorbed radioactivity measured in a thin-window gas-flow counter. Nucleic acid concentrations were based on the phosphorus content determined according to Fiske and Subba-Row. Protein was measured according to Lowry et al.5 Actinomycin concentrations were based on the absorption of a standard solution of 25 ,ug/ml, giving an O.D. (440 mu) of 0.358 in 0.01 Mll Tris pH 7.4 with 1 cm light path. The absorption of pure actinomycin is appreciably greater than this value which was established several years ago for a preparation containing inert material. DNA preparations: All DNA solutions were made in 0.01 ill Tris (pH 7.4) -0.01 M NaCl, except for crab dAT, which was dissolved in 0.75 III NaCl-0.05 M phosphate (pH 6.7). Highly polymerized calf thymus DNA was purchased from Sigma Chemical Co., St. Louis, 1Io.; DNA from Micrococcus lysodeikticus and Tetrahymena pyriformis was prepared by the method of Mfarmur,6 according to a modification suggested by N. Sueoka. Synthetic deoxyadenylic-deoxythymidylic (dAT) and deoxyguanylic-deoxycytidylic polymers (dGC), and crab dAT, were generously provided by A. Kornberg and N. Sueoka, respectively. Nonradioactive NTP was obtained from Pabst Laboratories and Sigma Chemical Co. Results.-Effect of actinomycin of NTP-PP32 exchange reaction: RNA-polymerase catalyzes a DNA-dependent exchange reaction between a single NTP and inorganic PP. This reaction is less sensitive to actinomycin than the synthetic reaction with the same DNA preparations1' I (Fig. 1). Nevertheless, the difference in actino-